Tissue-resident memory (TRM) cells are the main mediators of protective immunity to infections in peripheral tissues. Our recent published studies show evidence that this subset of T cells also mediates immunity to cancer. We found in our mouse model that the autoimmune destruction of melanocytes resulted in vitiligo, which led to the development of protective antigen-specific skin TRM cells. These cells provided long- lived protection against melanoma in the skin. While vitiligo was previously a rare phenomenon, now up to twenty-five percent of melanoma patients on immunotherapy will develop vitiligo, and those who do have been shown to have an improved progression-free and overall survival. We hypothesize that skin TRM cells underlie the durable tumor immunity in melanoma patients who develop vitiligo, and, by characterizing these cells, we can create a gene signature to predict patient immune response to melanoma. Our first Specific Aim will be to identify a TRM gene signature that is predictive of survival in vitiligo-affected melanoma patients. Our preliminary mouse data reveal that skin TRM cells from melanoma-associated vitiligo mice have a distinct gene signature, and this signature was shown to have a significant association with improved survival in melanoma patients. We will test this by defining the transcriptional signature of TRM cells from vitiligo-affected melanoma patients in Sub-Aim 1A. We will use single cell technology to define a global transcriptional profile of human melanoma-associated vitiligo skin TRM cells, and in Sub-Aim 1B, we will determine the significance of this human TRM transcriptional signature in predicting prognosis and patient sensitivity to immunotherapy. We will apply the TRM gene signature generated in Sub-Aim 1A to published gene-expression datasets from melanoma patients under standard treatment to associate resulting sample-specific TRM scores with disease prognosis. We will also apply this signature to several immunotherapy datasets to validate its relation with patient response to immune checkpoint blockade therapy. Finally, our second Specific Aim will determine if vitiligo- affected melanoma patient skin preserves TRM cells with broad tumor antigen specificity. This Aim will test the hypothesis that vitiligo-affected skin preserves a diverse repertoire of tumor-reactive TRM cells in humans. We will assess the TCR diversity, clonal abundance, and overlap in melanoma patient skin, with matched blood and tumor. If we demonstrate shared TCR clones in matched skin and tumors of these long-term melanoma survivors, this promising result will provide important insights into the basis of immunological memory against cancer. Overall, the studies in this proposal will not only bring us closer to understanding the intricate relationship between autoimmunity and tumor immunity, but will facilitate future clinical trials aimed at inducing durable cancer-fighting skin TRM cells in patients, thereby providing new avenues for immunotherapy development and improved cancer patient survival.